Abstract Collectively, the three RAS genes (HRAS, NRAS and KRAS) are the most mutated oncogenes in human cancers, and of these, KRAS is the isoform most frequently mutated (72%), accounting for ?90% of all RAS mutations in pancreatic, lung and colorectal tumors. Accordingly, there is intense interest in developing anti- RAS cancer therapies. RAS cycles between GDP-bound ?inactive? and GTP-bound ?active? states, and binds guanine nucleotides via a large central pocket surrounded by the dynamic ?switch? regions of the protein. Cancer-associated mutations in RAS isoforms invariably populate RAS with GTP thus rendering them constitutively activated. Virtually all current strategies which aim to find direct inhibitors of RAS are designed to compete with the binding of effectors, such as RAF and PI3K. Unfortunately, the effector binding site on RAS is devoid of targetable pockets and generating molecules that bind with sufficient affinity (to make them useful as potential RAS chemotherapies) has proven difficult. However, two different groups have recently succeeded in developing allosteric inhibitors of RASG12C which irreversibly bind to the substituted cysteine side chain. This strategy of specifically targeting mutant forms of RAS may be more advantageous as inhibiting oncogenic RAS directly would seemingly be more efficacious while potentially offering less normal cell toxicity. While this discovery represents a proof of concept, it cannot be extended to other RAS proteins lacking the appropriately substituted reactive sidechains. One seemingly logical approach to inhibiting RAS signaling would be to develop reversible GTP- competitive inhibitors that block GTP binding to render RAS inactive. This approach is considered not possible by many because of the high affinity (picomolar) of RAS for GTP and the high concentration of guanine nucleotides in cells. However, we have recently shown that some RAS mutants exhibit a reduced ability to bind GTP, which paradoxically makes them oncogenic. These include RASG13D, RASA146T and RASK117N and account for ~30% of all mutant KRAS in colorectal cancers. Reduced affinity for GTP renders these RAS mutants vulnerable to small molecule inhibition with potential selectivity over normal RAS. Thus, we propose using our novel, newly developed fluorescence-based guanine nucleotide displacement assay in a high- throughput screening (HTS) program to search for inhibitors of oncogenic RAS.